Abstract The emergence of antibiotic-resistant bacteria coupled with the dwindling supply of new antibacterial therapeutics has created a medical crisis. Traditional industry-driven approaches that target the active sites of essential enzymes have begun to stall, yielding fewer new antibacterial agents than are needed to combat the alarming wave of drug-resistant pathogens that have become commonplace in clinical settings. Novel approaches to therapeutic discovery are essential for generating effective treatments against emerging bacterial threats. This proposal tests the utility of protein interfaces, rather than enzyme active sites, as targets for antibacterial drug development. This mode of action takes advantage of the essential nature of protein interactions in supporting cellular processes, which are underexplored therapeutic targets. With its many essential protein interactions, the bacterial DNA replication machinery is an ideal system that will be used to test the robustness of protein interfaces as antibacterial therapeutic targets. High-throughput screens will identify compounds that disrupt bacterial DNA replication protein complexes and the mechanisms of action of the compounds will be determined using a combination of structural, biochemical and cellular studies. Antibiotic activities of the compounds will be assessed with a broad spectrum of bacterial species. The proposed approach will simultaneously test the extent to which protein interfaces can be used for antibacterial drug development and the suitability of DNA replication protein complexes as direct targets for such inhibitors. The rewards of this proposed research could pave the way to much needed antibacterial lead compounds and establish protein interfaces as novel targets for antibacterial drug development.